Hemostatic tone is dynamically established as the net balance between ongoing procoagulant versus anticoagulant and fibrinolytic processes. Antithrombin (AT) is a major anticoagulant that slowly neutralizes proteases of the coagulation cascade through the formation of 1:1 enzyme•AT complexes. The rate of neutralization is dramatically enhanced by heparin, a variant of heparan sulfate (HS) from mast cells. It has been hypothesized that heparan sulfate proteoglycans (HSPGs) on the endothelial cell surface might similarly accelerate AT activity and thereby contribute to the nonthrombogenic properties of blood vessels (Damus, et al. (1973 Nature 246:355–357). Indeed, the perfusion of purified thrombin (T) and AT into the hind limbs of rodents led to an elevated rate of T•AT complex formation that was HS-dependent. Endothelial cells produce only a small subpopulation of anticoagulant heparan sulfate (HSact) that binds AT and accelerates in vitro T•AT complex generation (Rosenberg, et al. (1997) J. Clin. Invest. 99:2062–2070; Rosenberg (2001) Thromb. Haemost. 86:41–50). This property distinguishes HSact from the bulk of endothelially generated heparan sulfate (HSinact), which lacks in vitro anticoagulant activity. However, it is unclear whether HSact is a major physiologic modulator of hemostasis.
For major hemostatic regulators, changes in the activity level can result in a hypercoagulable state (Rosenberg (2001) supra; Thomas and Roberts (1997) Ann. Intern. Med. 126:638–644; Hogan, et al. (2002) Thromb. Haemost. 87:563–574). For example, mutations that reduce the level of AT activity primarily predispose patients to venous thrombosis. Complete AT deficiency appears incompatible with human life, and in mice causes intrauterine death from an extreme hypercoagulable state, consumptive coagulopathy (van Boven and Lane (1997) Semin. Hematol. 34:188–204; Ishiguro, et al. (2000) J. Clin. Invest. 106:873–878). Yet, the contribution of HSact deficiency towards human hypercoagulable states is unknown.
Modulation of HSact levels requires knowledge of HSact structure and biogenesis. HS and heparin are functionally diverse biopolymers that occur on specific core proteins as a repeated disaccharide unit (N-acetylglucosamine α1->4 hexuronic acid β1->4) that is partially decorated with N- and O-sulfate groups. The specific arrangement of these substituents gives rise to distinct binding motifs that activate an array of important biologic effector molecules. Such structures arise through remodeling of the copolymer backbone by a relatively ordered series of reactions involving four families of sulfotransferases (Iozzo (2001) J. Clin. Invest. 108:165–167; Esko and Lindahl (2001) J. Clin. Invest. 108:169–173). For AT, the minimum binding domain generated in HSact and heparin is the pentasaccharide: ->N-acetylglucosamine 6-O-sulfate->glucuronic acid->glucosamine N-sulfate 3-O-sulfate±6-O-sulfate->iduronic acid 2-O-sulfate->glucosamine N-sulfate 6-O-sulfate->. AT forms specific contacts with several moieties; however, the central 3-O-sulfate group is absolutely essential for both high affinity binding and enhancement of AT activity (Rosenberg, et al. (1997) supra). 3-O-sulfates are the rarest of HS modifications, typically comprising <0.5% of total sulfate moieties (Shworak, et al. (1994) J. Biol. Chem. 269:24941–24952; Colliec-Jouault, et al. (1994) J. Biol. Chem. 269:24953–24958), suggesting a potential regulatory role.
The regulation of HSact production has been elucidated over the past decade. Core proteins appear to exert minimal influence, as a single core can bear either HSact or HSinact (Shworak, et al. (1994) supra). Instead, HSact results from a discrete biosynthetic pathway regulated by a limiting biosynthetic factor (Shworak, et al. (1994) supra; Colliec-Jouault, et al. (1994) supra). Establishment of conditions for cell-free synthesis of HSact revealed a limiting activity that modifies only a portion of potential precursors, thereby defining cellular production of HSact (Shworak, et al. (1996) J. Biol. Chem. 271:27063–27071). The critical enzyme was purified, cloned and identified as the long sought heparan sulfate 3-O-sulfotransferase-1 (also known as heparin-glucosamine 3-O-sulfotransferase, 3-OST-1) (Liu, et al. (1996) J. Biol. Chem. 271:27072–27082; Shworak, et al. (1997) J. Biol. Chem. 272:28008–28019; WO 99/22005). 3-OST-1 preferentially modifies selected HS structures to create the minimum binding domain pentasaccharide. 3-OST-1 also creates a limited range of 3-O-sulfated structures that do not bind AT (Zhang, et al. (2001) J. Biol. Chem. 276:28806–28813), but the biologic relevance of these structures is unknown. To date, 3-OST-1 has only been implicated in regulating hemostatic tone.
Four additional 3-OST isoforms have been isolated, but these isoforms have dramatically distinct substrate preferences; therefore they may regulate distinct biologic properties of HS (Shworak, et al. (1999) J. Biol. Chem. 274:5170–5184; Liu, et al. (1999) J. Biol. Chem. 274:5185–5192; Shukla, et al. (1999) Cell 99:13–22). Some of these isoforms can generate HSact, but at about 250-fold lower efficiency than 3-OST-1 (Yabe, et al. (2001) Biochem. J. 359:235–241). Thus, 3-OST-1 appears to be the dominant isoform regulating in vivo HSact production. Moreover, selective regulation stems from the enzymatic specificity of 3-OST-1 and the paucity of 3-O-sulfates within HS.
U.S. Patent Application No. 20020022255 provides transgenic mice containing sulfotransferase gene disruptions and methods of screening such animals for identification of drugs, pharmaceuticals, therapies, and interventions which may be effective in treating a disease or other phenotypic characteristics of the animal. Specifically provided are phenol/aryl forms of sulfotransferases which, when deleted in a transgenic animal result in behavioral phenotypes of aggression, hyperactivity, increased activity or decreased anxiety.
It has now been found that mice selected for lacking 3-OST-1 activity exhibit characteristics associated with myxomatous valvular disease.